PROJECT SUMMARY Children with Down syndrome (DS) are at substantial risk of developing acute leukemia, which suggests that there are leukemia promoting genes on chromosome 21. Chromatin Assembly Factor 1B (CHAF1B), which resides in the DS critical region, is a member of the heterotrimeric CAF1 chromatin assembly complex that is responsible for depositing H3/H4 heterodimers at the replication fork during S-phase. CHAF1B levels are elevated in DS-AML patient samples, as well as non-DS AML cell lines and primary AML samples when compared to healthy samples. While elevated expression is associated with poor prognosis in most tumors, the mechanism by which CHAF1B promotes leukemogenesis is unknown. My preliminary data show that CHAF1B directly binds discrete regions of chromatin associated with promoters and enhancers of myeloid differentiation genes. Deletion of Chaf1b in myeloid leukemia cells results in replacement of CHAF1B on the chromatin with the pro-differentiation transcription factors including CEBPA and the subsequent activation of a myeloid differentiation transcriptional program. Therefore, I propose that CHAF1B is required to maintain the undifferentiated state of MLL-AF9 leukemic cells through a role as a transcriptional regulator. In this proposal, I will study the mechanism of CHAF1B-dependent maintenance of leukemic stem cells. The central hypothesis is CHAF1B maintains the undifferentiated state of myeloid leukemic cells by competing for chromatin occupancy with transcription factors at the promoters and enhancers of differentiation genes. This competition leads to reduced expression of these genes and the resulting maintenance of the leukemic blast phenotype. In Aim 1, I will explore the mechanism of how the different domains of the CHAF1B protein contribute to maintaining the undifferentiated state of myeloid leukemic cells. Then, in Aim 2, I will establish CHAF1B as a therapeutic target in AML (including DS-AML) by determining its contribution to the progression of hematologic tumors in vivo and primary human patient samples in vitro. My ultimate goal is to use the information gained during the K99/R00 award to develop novel small molecule compounds that can block CHAF1B function and control AML tumors in vivo by differentiation. My career goal is to become an independent investigator and leader in the field of chromatin assembly and its effects on hematopoietic malignancies with the ultimate goal of developing new therapeutic strategies to treat leukemia. During the mentored K99 phase, my main technical goal is to become proficient at analysis of large-scale NGS data sets and basic proteomics. The impact of the K99 phase will be enhanced by my collaborations with Dr. Scott Armstrong at Dana Farber Cancer Center at Harvard University and Dr. Yubin Ge at Wayne State University, by allowing me to expand my training outside my home institution of Northwestern University. Dr. John Crispino and my advisory team will guide me through the completion of my postdoctoral training and assist me with my transition to independence.